This invention relates to neurotrophic factors and their involvement in neurodegenerative and other neuropathic disorders. More particularly, this invention relates to analogs of nerve growth factor.
1. Field of the Invention
Nerve growth factor (NGF) and other growth factors act via cell surface receptors to prevent neuronal death in the contexts of neural development, neurodegenerative disease, ischemia, axotomy and excessive excitatory amino acids (reviewed in: Longo et al., Nerve Growth Factor actions in the PNS and CNS. Eds: S. E. Loughlin and J. H. Fallon, Academic Press; pp 209-256 (1993)). They also promote neural regeneration and enhance neuronal function. The sequence of murine NGF and human NGF is reported in Ullrich et al. Nature 303:821-825 (1983).
Therapeutic applications of native neurotrophic factors have been limited by poor ligand penetration of the blood-nerve barrier, blood-brain barrier and CNS parenchyma, ligand stability and receptor specificity, attributable in part to the size of the native factors (Friden et al., "Blood-brain barrier penetration and in vivo activity of an NGF conjugate", Science, 259:373-377 (1993)). One approach for overcoming these limitations consists of development of low molecular weight, stable agonists or partial agonists capable of mimicking specific activities of native neurotrophic factor ligands.
Very few agents have been discovered which mimic both the survival- and neurite-promoting neurotrophic activity of NGF. Phorbol esters mimic NGF and ciliary neurotrophic factor (CNTF) neurotrophic activity presumably by influencing protein kinase C signal transduction (Montz et al., "Tumor-promoting phorbol diester mimics two distinct neuronotrophic factors", Dev. Brain Res, 23:150-154 (1985)). Gangliosides and several structurally unrelated lipids and detergents promoted survival and neurite outgrowth of dissociated sympathetic, ciliary and dorsal root ganglion (DRG) neurons and survival of PC12 cells, although this activity was unlikely to be mediated by specific NGF receptor interactions (Ferrari et al., "Gangliosides rescue neuronal cells from death after trophic factor deprivation", J. Neurosci. 13: 1879-1887 (1985)). Bay et al. ("Assessment of neurotrophic activity of a broad spectrum of pharmacologic agents in PC12 cells", Soc. Neurosci. Abst. 16:995 (1990)), assessed 65 synthetic and naturally occurring compounds for NGF-like neurotrophic activity with PC12 cells and found that, except for other growth factors, only cyclic AMP analogs had trophic activity. Triap (1,1,3 tricyano-2-amino-1-propene) is a small molecule which promoted survival and neurite outgrowth of cultured sympathetic ganglion neurons and PC12 cells (Paul et al., "1,1,3 tricyano-2-amino-1-propene (Triap) stimulates choline acetyltransferase activity in vitro and in vivo." Dev. Brain Res. 67:113-120 (1992)). The observations that Triap maximally induced ChAT enzyme activity at concentrations that did not affect neuronal morphology and that Triap was synergistic rather than additive to NGF in promoting ChAT activity led these investigators to suggest that Triap did not act through NGF receptors. Lehmann et al., ("Neurite outgrowth of neurons of rat dorsal root ganglia induced by new neurotrophic substances with guanidine group." Neurosci. Lett. 152:57-60 (1993)), reported that two of 19 compounds related to isaxonine (2-isopropylamino-pyrimidine) were particularly effective in promoting neurite outgrowth from DRG explants. That these compounds acted by mechanisms separate from or in addition to those related to NGF was also suggested by the observation that the effect of these compounds was additive to NGF even at optimal NGF concentrations. No NGF agonists or partial agonists (agents known to act as NGF ligands at NGF receptors) have been described.
Several approaches have been used to deduce which domains of the NGF molecule interact with NGF receptors. Synthetic peptides with sequences corresponding to four regions of NGF have been tested for their ability to inhibit NGF neurotrophic activity. Peptides corresponding to NGF region 28-38 inhibited (i.e., they were NGF antagonists), in a sequence-specific manner, NGF neurotrophic effects on DRG neurons, but not those of CNTF or phorbol ester (Longo et al., "The in vitro biological effect of nerve growth factor is inhibited by synthetic peptides", Cell Regulation 1:189-195 (1990)). Subsequent NGF crystallography studies revealed that NGF contained three hydrophilic .beta.-hairpin loops which were situated at NGF's surface and thus constituted likely candidates for receptor interaction sites (McDonald et al., "New protein fold revealed by a 2.3-A resolution crystal structure of nerve growth factor." Nature 354:411-414 (1991)). One of these .beta.-hairpin loops was formed by the NGF 29-35 region. Studies from two additional laboratories have confirmed that region 29-35 peptides can inhibit NGF activity in a sequence-specific manner (Ross et al, Soc. for Neurosci. Abstr., v20(1-2) p678, Abstr. 289.2 and 289.3 (1994); LeSauteur et al, "Small peptide mimics of nerve growth factor bind TrkA receptors and affect biological responses." J. Biol. Chem., 270:6564-6569 (1995)). Recombinant molecules containing modified residues in this region or consisting of various combinations of NGF and BDNF demonstrated that NGF region 25-36 along with other .beta.-hairpin loop and non-loop regions significantly influenced NGF/NGF-receptor interactions (Ibanez et al., "Chimeric molecules with multiple neurotrophic activities reveal structural elements determining the specificities of NGF and BDNF", EMBO J. 10:2105-2110 (1991); Suter et al., 1992; Ibanez et al., "Disruption of the low affinity receptor-binding site in NGF allows neuronal survival and differentiation by binding to the trk gene product" Cell 69:329-341 (1992); Ibanez et al., "An extended surface of binding to Trk tyrosine kinase receptors in NGF and BDNF allows the engineering of a multifunctional pan-neurotrophin", EMBO J. 12:2281-2293 (1993), Ilag et al., "Role of variable .beta.-hairpin loop in determining biological specificities in neurotrophin family", J. Biol. Chem. 269:19941-19946 (1994)). U.S. Pat. No. 5,349,055 describes NGF mutants in the .beta.-hairpin loop regions 25-36 and 95 which have reduced binding to p75.sup.LNGFR (low affinity NGF receptor). WO 90/10644 describes polypeptides corresponding to regions 90-103 and 23-35 of native NGF and their use to raise antibodies to NGF. EP 0 476 933 A1 discloses cyclic peptide analogs of HCNP (Hippocampal Cholinergic Neurotrophic Peptide), but no NGF agonist activity was described. Polyclonal antibodies raised against individual NGF .beta.-hairpin loop peptides also indicated that multiple domains influence NGF function (Murphy et al., "Immunological relationships of NGF, BDNF, and NT-3: Recognition and functional inhibition by antibodies to NGF", J. Neurosci., 13:2853-2862 (1993)).
The complexity of NGF/NGF-receptor interactions has been further described by McDonald and Chao, "Structural determinants of neurotrophic action", J. Biol. Chem. 270:19669-19672 (1995). NGF interacts with cell surface low affinity (p75.sup.LNGFR, referred to as p75) and high affinity (p140.sup.trk, referred to as Trk) receptors. In one model for signal generation by NGF, p75 is required for formation of high-affinity NGF binding sites via p75-Trk heterodimers (Mahadeo et al., "High affinity nerve growth factor binding displays a faster rate of association than p140.sup.trk binding", J. Biol. Chem. 269:6884-6891 (1994)). In another model, Trk is capable of functioning independently (Clary et al., "TrkA cross-linking mimics neuronal responses to nerve growth factor", Mol. Biol. of the Cell 5:549-563 (1994)), although p75 can modulate NGF-induced Trk activity (Verdi et al., "p75.sup.LNGFR regulates Trk signal transduction and NGF-induced neuronal differentiation in MAH cells", Neuron 12: 733-745 (1994)). The existence of NGF dimers (Angeletti et al., "Subunit structure and amino acid composition of mouse submaxillary gland nerve growth factor", Biochem. 10:463-469 (1971)) is compatible with models in which NGF activates receptors by mediating formation of receptor homo- or heterodimers. Potential p75 functions which may occur independently of Trk include mediation of NGF-stimulated invasion of extracellular matrix by melanoma cells (Herrmann et al, "Mediation of NGF-stimulated extracellular matrix invasion by the human melanoma low-affinity p75 neurotrophin receptor: Melanoma p75 functions independently of trkA", Mol. Biol. of the Cell 4:1205-1216 (1993)), NGF-mediated inhibition of apoptosis, (Taglialatela et al, "Effect of p140trkA suppression on NGF and BDNF rescue of apoptotic PC12 cells", Soc. Neurosci. Abst. 21:1061 (1995)) and induction of sphingomyelin hydrolysis (Dobrowsky et al, "Activation of the sphingomyelin cycle through the low-affinity neurotrophin receptor", Science, 265:1596-1599 (1994)).
Despite this degree of ligand-receptor complexity, and despite the fact that previous peptide analogs of NGF have been NGF antagonists, it has been surprisingly discovered that a class of analogs of nerve growth factor amino acid sequences have NGF agonist activity.
2. Summary of Related Art
Small Peptide Mimics of Nerve Growth Factor Bind TrkA Receptors and Affect Biological Responses; L LeSauteur, L. Wei, B. F. Gibbs, and H. U. Saragovi; The Journal of Biological Chemistry, 270:6564-6569, (1995); described cyclic NGF antagonist peptides derived from the .beta.-turn hairpin loop of NGF.
The In Vitro Biological Effect of Nerve Growth-Factor Is Inhibited by Synthetic Peptides; F. M. Longo, T. H. Vu, and W. C. Mobley; Cell Regulation, 1:189-195, (1990); described NGF antagonist peptides from the mouse NGF region 26-40.
Conformationally Constrained Peptides Block NGF binding; G. M. Ross et al.; 24th Ann. Mtq. Soc. Neurosci., Soc. for Neurosci. Abs., 20:1-2, Abstract 289.2 (1994); describes conformationally constrained peptide antagonists derived from NGF.
Peptide Mimics of an NGF Domain Inhibit Kindling and Neuronal Sprouting in Rats; K. Rashid, C. E. E. M. Van der Zee, G. M. Ross, C. A. Chapman, R. J. Racine, R. J. Riopelle, and M. Fahnestock; 24th Ann. Mtq. Soc. Neurosci., 20:1-2 Abstract 289.3 (1994); describes bicyclic peptide NGF antagonists.
Neurotrophic Peptide Derivatives; European Patent No. 0 476 933 A1; Y. Ueki, N. Fukushima, Y. Okeda, T. Nishihara, K. Ono, and T. Irie, (1992); discloses neurotrophic cyclic disulfide bridged peptides related to Hippocampal Cholinergic Neurotrophic Peptide.